The Hormel Institute, University of Minnesota, Austin, MN, USA.
J Lipid Res. 2013 Apr;54(4):1103-13. doi: 10.1194/jlr.M034744. Epub 2013 Jan 31.
Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifically transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfluidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fluorescence data. At biomembrane-like packing (30-35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in curvature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negatively-charged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating the novel capabilities of the microfluidics-based film balance for investigating protein-membrane interfacial interactions.
在两性蛋白中，人类糖脂转移蛋白 (GLTP) 形成了一种结构独特的折叠，能够在膜上进行构象转换，从而特异性地转移糖脂。具有曲率诱导的包装应力的磷脂酰胆碱 (PC) 双层膜比非应激 PC 双层膜更能促进糖脂的囊泡间快速转移，这引发了关于平面细胞质侧生物膜是否适合 GLTP 相互作用的问题。在此，通过一种新的基于微流控的表面天平来评估 GLTP 介导的荧光糖脂（四甲基硼二吡咯甲川（BODIPY）标记）从脂质单层解吸的动力学，该表面天平在监测脂质层侧向堆积的同时，同时获取表面荧光数据。在类似于生物膜的堆积（30-35 mN/m）下，GLTP 从 POPC 单层中摄取 BODIPY-糖脂几乎不存在，但可以通过降低表面压力来诱导，使其与曲率胁迫双层膜的堆积一致。相比之下，1-棕榈酰基-2-油酰基-磷脂酰乙醇胺（POPE）基质在高和低表面压力下都能支持 GLTP 对 BODIPY-糖脂的摄取。出人意料的是，带负电荷的细胞质侧脂质，即磷脂酸和磷脂酰丝氨酸，在高表面压力下也支持 GLTP 摄取 BODIPY-糖脂。值得注意的是，在 POPC 中同时包含 1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸（5 mol%）和 POPE（15 mol%）能够在高表面压力下协同激活 GLTP。我们的研究表明，基质脂质头部基团组成，而不是分子堆积本身，是 GLTP 折叠功能的关键调节因子，同时展示了基于微流控的薄膜天平在研究蛋白-膜界面相互作用方面的新能力。
